Biomaterials that can physically control the fate of stem cells are critical for the application of in situ tissue engineering. However, the mechanisms underlying this phenomenon remain unclear. In this study, cellulose and silk composite substrates, known to induce physically the chondrogenic differentiation of human mesenchymal stem cells (MSCs), were regenerated to fabricate two-dimensional film surfaces. MSCs were grown on these surfaces in the presence of chemicals that interfere with the biochemical and mechanical signalling pathwys of MSCs. The data shows that preventing substrate surface elasticity transmission results in significant downregulation of chondrogenic gene expression. Interference with the classical, chondrogenic Smad2/3 Phosphorylation pathway does not impact the chondrogenic capacity of the substrates. The results highlight the importance of the substrate mechanical elasticity on chondrogenic MSCs and its independence of known chondrogenic biochemical pathways.